Many industrially important products are produced from members of the genus Bacilli. Some of these include proteases, amylases, and beta-glucanases (Priest, 1977, Bacteriological Review, 41: 711-753). Most of these enzymes are either produced by Bacilli after the initiation of sporulation or are timed with the onset of the stationary phase.
Bacillus subtilis I-168 is the most extensively studied member of the Bacilli and has a well developed genetic system as the result of many years of academic research. As a result, there is a considerable amount of knowledge pertaining to the expression of extracellular enzymes and many mutants of B. subtilis I-168 exist that are altered in the expression of these enzymes. There are several different known genes which code for proteins that regulate the expression of extracellular enzymes. Although the exact molecular mechanism of this regulation is not completely understood, it is now clear that the product of these genes interact either directly or indirectly at the transcriptional level of the target enzymes (Ferrari et al, 1988, J. Bacteriol. 170: 289-295; Henner et al, 1988, J. Bacteriol. 170: 296-300).
Regions of the DNA around promoters can actively interact with certain transcriptional control factors and effectively regulate the strength and efficiency of a particular promoter. This interaction will result either in a stimulation or in a repression of the transcription of that particular target gene. As a consequence, mutations in either the genes coding for these transcriptional control factors or in regions of the chromosome with which they interact are expected to yield more or less mRNA of the target gene. This in turn will result in higher or lower amounts of enzyme synthesized by the cell.
With the advent of genetic engineering techniques, it has been possible to clone and express a gene from another species in B. subtilis I-168 or other Bacillus species. Most of these studies have involved the use of multicopy plasmids as cloning vectors. Although the yield of the cloned protein was increased significantly over the production in the wild type host, it was far below the yield of enzyme reported for production strains obtained through mutagenesis. This was also the case when the enzyme was under the control of strong transcriptional and translational elements derived from B. subtilis. Apparently the production of enzyme obtained from the multicopy, replicating plasmids was not affected by the presence of certain mutations, i.e. sacU(Hy), sacQ(Hy) and hpr (Dedonder et al, Microbiology 1976, D. Schlessinger (Ed), pp 58-69, American Society for Microbiology, Washington, DC; Palva et al. 1983, FEMS Microbiology Letters, 17: 81-85; Ferrari, unpublished observation), described as being able to induce hyperproduction of several secreted enzymes. These regulatory genes are unlinked to the structural gene for the affected enzymes and therefore one should expect that they exert their stimulating activity to a greater degree also when the gene is present in a multicopy plasmid.
There are at least 6 different regulatory genes known to interact either directly or indirectly with the transcription of the subtilisin gene: sacU, sacQ, prtR, hpr, sin and abrB.
Certain mutations in the sacU gene encode for a modified polypeptide which has the most pleiotropic effect among all these known regulatory genes. A strain carrying for example sacU (Hy) mutations can sporulate in presence of high levels of glucose, has a very low efficiency of transformation, lacks flagella and it is also responsible for the hyperproduction of several secreted enzymes. (Dedonder et al, Microbiology 1976, see above).
The sacQ gene from three different bacilli has been cloned and characterized (Amory et al, 1987, J. Bacteriol, 169, 234; Yang et al, 1986, J. Bacteriol, 166, 133; and Tomioka et al, 1985, J. Biotechnol 3,85). The gene isolated from B. subtilis encodes a 46 amino acid polypeptide. The sacQ gene when hyperexpressed either because of a mutation in its promoter or when present in a multicopy plasmid causes hyper production of certain extracellular enzymes, including the alkaline protease (Yang et al, 1986, J. Bacteriol 166, 113-119).
The prtR gene encodes a 60 amino acid polypeptide. When this gene is present in B. subtilis in a multicopy plasmid it causes increased production of proteins. However, unlike for sacQ, there have been no chromosomal mutations to date for this gene which increases protease production (Yang et al., 1987, J. Bacteriol. 169, 434-437).
The hpr gene encodes a 203 amino acid protein and certain mutations in this gene stimulate alkaline protease production. It has been established that mutations which cause the loss of this gene product stimulate transcription of the subtilisin gene (Perego and Hoch, 1988, J. Bacteriol. 170, 2560-2567). The product of the hpr gene therefore is most likely a repressor of the subtilisin gene. It has also been determined that the scoC gene and possibly the catA gene are identical to the hpr. The notation hpr will be used thereafter to refer to any of the three alleles called hpr, scoC or catA.
The sin gene encodes a polypeptide of 111 amino acids (Gaur et al, 1986, J. Bacteriol, 168: 860-869). An insertional inactivation of the gene causes the production of a higher level of alkaline protease, suggesting that this gene also acts as a repressor of the expression of alkaline protease.
Finally the abrB gene codes for a polypeptide of 97 amino acids. Also for this gene there is evidence that it is a repressor of the transcription of the alkaline protease and might have an important role in the temporal regulation of this gene.